Microdissection and amplification of coding sequences from a chromosome fragment restoring male fertility in alloplasmic male-sterile tobacco.

An alloplasmic male-sterile line of tobacco, containing the nucleus of Nicotiana tabacum and the cytoplasm of Nicotiana repanda, is restored to fertility by introgression of an alien chromosome fragment obtained from the cytoplasm donor. To isolate the restorer gene(s), the alien chromosome fragment was microdissected from metaphase plates of the restored line. The microdissected chromosomes represented only 0.1 pg of DNA, which was amplified using a degenerate oligonucleotide-primed PCR method (DOP-PCR), from which a chromosome fragment specific library was created. Compared with previous strategies used for microcloning, a modified and improved method was developed by the subsequent isolation of expressed sequences. The library was screened with cDNA probes synthesized by reverse transcription and DOP-PCR amplification (RT/DOP-PCR), of total RNAs isolated from early developing restored and male-sterile flower buds. By this strategy, transcribed DNA sequences specific for the restored line were cloned.